新长链非编码RNA ORAOV1-B促进口腔黏膜癌变的机制及潜在的临床意义研究

Oral mucosal epithelial cells penetrate the basement membrane to invade deep tissue is the key to oral carcinogenesis. Understanding its mechanism and blocking its process is the frontier that is concerned. The applicant found a new long non-coding RNA and named it lncRNA ORAOV1-B, which binds to Hsp90 and regulates epithelial to mesenchymal transition (EMT) through NF-κB and TNFα. We hypothesized that Hsp90-NF-κB-TNFα-EMT pathway plays an important role in the process of epithelial cells breaking through the basement membrane. In order to verify this hypothesis, it is proposed to further explore the relationship between ORAOV1-B expression and epithelial cell breakthrough through the basement membrane in clinical samples of different stages of oral carcinogenesis and to verify its function at the cellular level; later to study the mechanism of ORAOV1-B regulating the migration and invasion of epithelial cells through Hsp90-NF-KB-TNFα-EMT signaling pathway. Finally, the oral mucosa carcinogenesis model induced by 4NQO drinking mice was genetically modified to confirm the in vitro findings. The study will not only help further elucidate the mechanism of oral carcinogenesis, but also support the role of lncRNA in the regulation of epithelial cells to break through the basement membrane, providing new ideas for seeking new targets and blocking oral mucosa carcinogenesis.

口腔黏膜上皮细胞突破基底膜入侵深层组织是口腔黏膜癌变的关键。了解其机理、阻断其过程是受关注的前沿。申请人前期研究发现一新长链非编码RNA命名为lncRNA ORAOV1-B，其结合Hsp90并可通过NF-κB和TNFα调控上皮间质转化（EMT）。我们假设其通过Hsp90-NF-κB-TNFα-EMT通路在上皮细胞突破基底膜的过程中发挥重要调控作用。为验证此假说，拟首先在口腔黏膜癌变不同阶段的临床样本中进一步探索ORAOV1-B表达与上皮细胞突破基底膜的关系，并在细胞水平验证其功能；之后解析ORAOV1-B通过Hsp90-NF-κB-TNFα-EMT信号元件调控该过程的作用机制；最后通过基因编辑小鼠4NQO饮水诱导口腔黏膜癌变模型，确认体外的研究结果。研究不仅有助于进一步阐明口腔黏膜癌变的机制，也可佐证lncRNA在调控上皮细胞突破基底膜中的作用，为寻求新靶点、阻断其癌变提供新思路。

口腔黏膜上皮细胞突破基底膜入侵深层组织是口腔黏膜癌变的关键。了解其机理、阻断其过程是受关注的前沿。课题负责人前期研究发现一新长链非编码RNA命名为lncRNA ORAOV1-B，结合前期研究发现提出科学假说lncRNA ORAOV1-B可能通过Hsp90-NF-κB-TNFα-EMT通路在上皮细胞突破基底膜的过程中发挥重要调控作用。为验证此假说，拟首先在口腔黏膜癌变不同阶段的临床样本中进一步探索ORAOV1-B表达与上皮细胞突破基底膜的关系，并在细胞水平验证其功能；之后解析ORAOV1-B通过Hsp90-NF-κB-TNFα-EMT信号元件调控该过程的作用机制；最后通过基因编辑小鼠4NQO饮水诱导口腔黏膜癌变模型，确认体外的研究结果。研究中课题组首先在多株HNSCC细胞中发现了lncRNA ORAOV1-B表达与淋巴结转移呈正相关关系，并在OSCC组织样本中发现lncRNA ORAOV1-B相对表达量及ORAOV1-B/ORAOV1比值与淋巴结转移的发生和严重程度正相关。利用人表达谱芯片结果及DAVID分析以及在ORAOV1-B组添加依那西普，证实TNFα-NF-κB环路下游EMT蛋白通路可能是lncRNA ORAOV1-B调控细胞侵袭迁移能力的关键信号通路。进一步利用RNA pull-down技术和液相色谱-串联质谱法证实lncRNA ORAOV1-B通过直接结合Hsp90间接上调NF-κB-TNFα环路的信号传导。4NQO饮水诱导口腔黏膜癌变模型证实了lncRNA ORAOV1-B可上调癌变黏膜组织中TNFα的表达；裸鼠尾静脉注射OSCC细胞转移模型证实了lncRNA ORAOV1-B可促近肿瘤转移。研究从细胞侵袭迁移能力的角度进一步阐释了口腔黏膜癌变的机制，也佐证了lncRNA在调控上皮细胞突破基底膜中的作用，为寻求新靶点、阻断其癌变提供新思路。